KR20070078925A - Sample analysis method using microchip - Google Patents

Abstract

A sample analysis method using a microchip is provided to have superior detection sensitivity and detect a cell with minimum volume by preventing loss of a signal due to diffusion. A sample analysis method using a microchip by injecting a specimen(2) through a capillary tube tip(1) into a channel of a microchip forming the channel detecting a fluorescence method or an electrochemical detection method comprises steps of sequentially injecting a first ionic liquid(4) unmixed with water when injecting the specimen, a mixed liquid of electrolyte solution and the specimen, and a second ionic liquid unmixed with the water by using an electric field.

Description

Sample analysis method using microchip {SAMPLE ANALYSIS METHOD USING MICROCHIP}

1 is a view showing the injection of an ionic liquid and a very small volume of an aqueous sample, which is not mixed with water, in a tapered capillary tip, according to the present invention.

2 (a) and 2 (b) are schematic diagrams and photographs showing a sample injection device according to the present invention, respectively.

3 is an enlarged photograph of the connection portion between the microchip substrate and the capillary tip in the sample injection device shown in FIG.

4 and 5 are process charts showing a method of manufacturing a microchip substrate and a capillary tip, respectively.

Figure 6 is a schematic diagram showing the state of the material of the capillary inner wall after coating the inner wall of the capillary tip with a polymer.

7 and 8 are photographs of the results of injecting a sample of 300 pL and 30 pL volume into the sample injection device using an electric field, respectively.

9 is a photograph of a multi-segment sample injection result using the sample injection device according to the present invention.

10 is a comparison result of argon ion laser induced fluorescence signal of riboflavin (riboflavine) according to the use of the ionic liquid of the sample injected using the sample injection device of the present invention.

<Description of the symbols for the main parts of the drawings>

1: laser tapered molten silica capillary tip

2: sample filled in capillary

3: buffer solution filled in capillary tube

4: ionic liquid filled in capillary

5: chip substrate

6: interface

7: buffer solution reservoir

8: microchip channel

The present invention relates to a sample injection analysis method using a microchip.

Advances in science and technology enable the analysis of trace amounts, and there is a growing interest in technologies that handle very small volumes of samples or reagents. In particular, recent advances in synthetic chemistry and life sciences have led to an increase in target materials that need to be analyzed in areas such as new drug development and diagnostics.As a result, a large amount of expensive reagents or samples are required. The need for

Lab-on-a-chip technology has come to the fore in the face of an increasing share of handling very small amounts of samples and reagents. Lab-on-a-Chip is a chemical microprocessor that integrates several devices on several cm ² chips using photolithography or micromachining technology, which is widely used in the semiconductor industry. Automated experiments are possible.

Lab-on-a-chip is mainly implemented on glass, silicon, and plastic substrates, but many chips using glass were made in the early days. Recently, however, the transition to plastic chips suitable for mass production of reproducible, standardized chips is fast progressing. As a material for the plastic chip, polymer materials such as poly (dimethylsiloxane), PDMS, polymethylmethacrylate (PMMA), and polycarbonate (PC) are used. Among them, PDMS is the most widely used plastic material because of its excellent optical transmittance and easy molding.

Lab-on-a-chip, which has gained popularity recently, is expanding its range of applications, including high-speed separation and analysis of chemicals, environmental monitoring, analysis of pharmaceutical substances, and development of military applications. The most widely used uses are for high speed / high performance separation and analysis of compounds / medical substances. An important point in the separation experiment is to increase the separation efficiency, which depends a lot on the shape of the injected sample plug. Therefore, sample injection is one of the most important processes in separation / analysis.

Recently, many studies on living single cell analysis have been conducted in the research of life sciences, and since the lab-on-a-chip is similar in size of cells, many researches using this have been done.

One of the conventional methods of injecting a very small amount of sample into a lab-on-a-chip channel is to use an electric field. It controls the flow of the solution by applying voltage across the tiny channel filled with the solution, and injects the sample using the geometry of the microchannel, which allows separation / analysis using capillary electrophoresis on the chip. Most commonly used. Gated mode or pinched mode is used for microchip separation / analysis, in which microscopic samples are injected by adjusting electric field strength in channels such as double T and cross. It is used. This method is the most widely used method for lab-on-a-chip because it is relatively easy and simple, but it is difficult because the flow of the solution is greatly influenced by physical properties such as acidity, ionic strength, and viscosity of the solution to be injected. In addition, this method is a method that flows only a certain amount of the flow to the separation channel while flowing a solution, it is very expensive to waste a lot of samples, and the devices for manipulating the fluid has a complex disadvantage. And it is very difficult to set the experimental condition because it is affected by the difference in water level, and only one type of sample can be injected.

In addition, there is a method of injecting a very small amount of sample into the separation channel using pressure in addition to the method using the electroosmotic flow. However, it is limited in application because it cannot be used for capillary electrophoresis separation.

Recently, a method of mixing the above two methods has been proposed. Sample loading can be done using water pressure and separation using an electric field to prevent electroporation in single cell studies and reduce the chance of cells sticking to the surface in the channel. However, the method is complicated and difficult to operate, and has a fatal disadvantage that leakage to the separation channel occurs when the sample is injected.

In addition, there is a method using a sipper chip developed by Caliper. This is a method of vertically attaching the capillary to the bottom of the chip and then quantitatively injecting a sample from the well into the microchannel using a vacuum. However, this method has been made for a specific use for protein kinase analysis, so that various applications are difficult and chip manufacturing is difficult.

In addition, many studies have been carried out for the analysis of trace amounts of samples, but most of the time the signal is obscured by the spread of the sample is difficult to analyze the sample, the biological material in the living cells, tissues or organs selectively No injection analysis technique has been reported that can directly sample traces of traces.

The object of the present invention is to prevent the loss of signal due to diffusion to provide the best detection sensitivity and provide a high theoretical singular, and to detect the presence of traces of small volume of cells such as single cell analysis, especially in life science research. The present invention provides a method for analyzing a sample using a microchip, which is useful for detecting various samples, and injecting multi-segment samples with the same tip.

In order to achieve the above object, in the present invention, a method of analyzing a sample by injecting a sample into a channel through a capillary tip into a microchip in which a channel is formed and detecting it by a fluorescence method or an electrochemical method. And a first ionic liquid, a mixed solution of an electrolyte solution and a sample, and a second ionic liquid not mixed with water are sequentially injected using an electric field.

Hereinafter, the present invention will be described in detail.

The sample analysis method according to the present invention prevents the loss of the signal due to diffusion by injecting the ionic liquid using an electric field before and after sample injection, thereby providing excellent detection sensitivity and providing a high theoretical singular, so that the substance exists in trace concentrations. And in particular, in life science research, it is useful not only for the detection of very small volumes of cells such as single cell analysis, but also for multi-segment sample injection with the same tip.

The ionic liquid used in the present invention is a hydrophobic liquid which is not mixed with water, and cationic ionic liquids such as alkylimidazolium or N-alkylpyridinium, hexafluorophosphate and bis (trifluoromethylsulfonyl) And anionic liquids such as imide, trifluoromethylsulfonate or tetrafluoroborate. The alkylimidazolium and N-alkylpyridinium in the cationic ionic liquid preferably has an alkyl group of 2 to 12 carbon atoms. For example, alkylimidazolium is 1-butyl-3-methyl-imidazolium bis (trifluoromethylsulfonyl) imide, 1-butyl-3-methyl-imidazolium hexafluorophosphate, 1- Ethyl-3-methyl-methylimidazolium bis (trifluoromethylsulfonyl) imide, 1-ethyl-3-methyl-methylimidazolium tetrafluoroborate, 1-ethyl-2,3-methyl-imi Dazolium trifluoromethanesulfonate and 1-butyl-2,3-methyl-imidazolium hexafluorophosphate, wherein N-alkylpyridinium is N-butyl-pyridinium trifluoromethanesulfonate, 3-methyl-N-butyl-pyridinium hexafluorophosphate etc. are mentioned.

Samples that can be analyzed according to the present invention include neurotransmitters, DNA, RNA, proteins, enzymes, metabolite components, sugars, inorganic ions, etc. as various bioactive substances in cells, tissues and organs. Trace and expensive natural materials or environmental analytical samples are also included, but are not limited to these.

Preferred sample injection apparatus for microchip analysis of the present invention will be described in detail with reference to the accompanying drawings, but this is not intended to limit the scope of the present invention is presented by way of example only.

First, Figure 1 is a segment sample injection device for microchip analysis of the present invention, the ionic liquid 4 and the aqueous solution sample (2) which is not mixed with water by an electric field in the capillary tip (1) for taking a sample Show segment injection.

Figure 2 (a) and (b) is a schematic diagram showing a sample injection device and a photograph of the actual manufactured device. The sample injection device according to the present invention comprises a microchip substrate 5 having a channel 8 formed thereon, a capillary tip 1 for collecting a sample, and an interface 6 therebetween. The channels formed on the microchip substrate may be made into microchannels by any one of etching, molding, pressing, machining, laser processing, etc., and both ends of the buffer reservoir 7 and the capillary tip ( Connected to 1).

The microchip substrate used in the present invention may be a rubber, silicone rubber, plastic, metal, glass, or silica commonly used, and polydimethylsiloxane (PDMS) may be used as a material used for plastic chips in general. have.

The capillary tip used in the present invention may be made of rubber, silicon-based rubber, plastic, metal, glass, or silica, and in the case of using glass or silica, the capillary tip may be manufactured by tapering into a needle shape using a carbon dioxide laser. .

Figure 3 is an enlarged picture of the interface between the microchip and the capillary tip, interfacing can be achieved by placing an organic solvent in the chip having a chip width of 80% of the capillary diameter to expand the channel and then position and fix the capillary tip have.

4 shows a typical process ((a) to (f)) of manufacturing a microchip substrate for a lab-on-a-chip according to the present invention, and in FIG. 5, a capillary tip according to the present invention is manufactured ((a) to ( d)) is shown.

Referring to FIG. 4, in order to manufacture a microchip substrate for a lab-on-a-chip, first, a negative photoregist is coated on a silicon wafer (step (a)). Rubber, silicon-based rubber, plastic, glass, silica, etc. may be used as the material of the wafer, and a negative photosensitive layer may be a commercially available one, for example, SU-8 (Microchem.), AL-217 ( Aldrich) or KTFR (Kodak). Coating methods include spin coating or dip coating, preferably spin coating. In addition, the number of coating is not limited, but is preferably 1 to 2 times.

After the negative photoregist is coated on the silicon wafer, the photomask is covered thereon and exposed to ultraviolet light (step (b)). When the exposed silicon wafer is developed, an embossed mold having a desired pattern is obtained (step (c)).

A prepolymer such as PDMS is applied to a silicon wafer on which an embossed frame is formed to cast a top plate and cured (step d), and then peeled off the silicon wafer (step (e)).

The top plate replicated as described above is oxidized by surface treatment with a corona discharge formed using Tesla coil, and then fixed with a flat bottom plate such as a glass plate or a silicon wafer, whose surface is cleanly cleaned, and an adhesive (step (f) )) Complete microchip substrate for lab-on-a-chip with sample injection tip holder.

In the capillary tip manufacturing process shown in FIG. 5, first, a fused silica rod (step (a)) having a capillary tube formed therein is pulled at both ends in a molten state by a carbon dioxide laser using a micropipette puller. After tapering (step (b)), the tapered portion is cut with a cutter and polished for 30 minutes with a 0.3 μm alumina plate using a micro pipette beveler. The obtained capillary tip may be cut to a certain length, and the angled part may be ground with a soft sandpaper, and then (in step (c)), and then internally coated with a cationic polymer, if necessary, which may be used as a microchip substrate (5). (Step (d)).

The cationic polymer coating can be formed by electroosmotic flow by immersing both ends of the channel and tip in a buffer solution containing 0.5% polymer and applying a constant electric field to both ends, and shows the state of the material in the inner wall of the capillary tip after coating. As shown. Cationic polymer coating is for injecting ionic liquid into capillary tip and channel under electric field. After cationic polymer coating, the electroosmotic flow of the solution and the flow direction of ionic liquid under the electric field coincide to introduce the sample into the capillary tip. can do. Cationic polymers used in the present invention include polydimethyldiallyl ammonium chloride (PDDAC), polybrene (PB), polyethyleneimine (PEI), poly (methoxyethoxyethyl) ethylimine (PolyEG ₂ ) or polylysine (PL). ).

The method of analyzing a sample using a segment sample injection device for a lab-on-a-chip manufactured according to the present invention is as follows (see FIGS. 1 and 2 (a)).

First, the buffer is filled in the buffer reservoir 7 on the microchip, and a platinum electrode is put in the buffer reservoir to apply voltage. At this time, the voltage and time can be adjusted according to the diameter of the capillary tip and the sampling volume. For example, the inner diameter of the capillary or channel 3 is 20-100 μm and the capillary length is 1-5 cm. In this case, an electric field in the range of 1 to 600 V / cm may be applied for 0.1 second to 5 minutes. In addition, the capillary tip 1 is immersed in an ionic liquid, and then a platinum electrode is put in the ground to apply a voltage to inject the ionic liquid 4 into the capillary tip. By sequentially dipping the capillary tip portion into the liquid 4 to generate an electric field, a plurality of samples may be segmented into the capillary tip in such a manner that the ionic liquid and the sample-containing mixed solution are alternately repeated. At this time, when the first ionic liquid is injected, it is necessary to apply a voltage for a longer time than when the second ionic liquid is injected, because the smaller the diameter of the inlet portion of the capillary tip has a higher resistance.

As described above, according to the sample analysis method according to the present invention, the ionic liquid is injected by using an electric field before and after sample injection to prevent the loss of the signal due to diffusion to the maximum, thereby providing excellent detection sensitivity and providing a high theoretical number of fractions. It is useful for detecting a variety of samples including concentrations of substances and in particular microvolume cells such as single cell analysis in life science research, as well as multi-segment sample injection with the same tip and no diffusion of samples If the preparation time is long, the sample can be stored for some time.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example

According to the present invention, a boron using a sample injection device composed of a microchip substrate of polydimethylsiloxane (PDMS) and a tapered molten silica capillary tip (outer diameter: 360 μm, inner diameter: 50 μm) having an inner diameter of the tip end of less than 10 μm It was applied to sampling experiments of liquid samples in acid-sodium hydroxide buffer solution.

First, a capillary tip of 45 mm in length and a length of 30 mm from the chip buffer reservoir to the end of the interfaced capillary tip was prepared. 0.1M NaOH solution was filled with a syringe pump on the finished chip, and then washed with 400 μL of distilled water after 15 minutes. Then, the channels were filled with 0.5% polydimethyldiallyl ammonium chloride (PDDAC) electrolyte solution, and both ends were immersed in 0.5% PDDAC buffer solution for 20 minutes and electrophoresed.

Then, after filling the buffer solution in the buffer reservoir on the microchip substrate, the tip tip was immersed in the buffer solution, and a platinum electrode was placed therein. On the other hand, the tip of the capillary tip was immersed in the order of the ionic liquid, the mixed solution of the electrolyte solution and the sample, and the ionic liquid, respectively, and each electrode was grounded with a platinum electrode. 0.1 μM riboflavin was used as the sample to be injected, and the ionic liquid was selected from 1-butyl-3-methylimidazolium bis (trifluoromethylsulfonyl) imide which was not mixed with water. In order to monitor the sample injection process of the clear liquid sample, the Sudan III reagent was dissolved in the ionic liquid to give a red color. Apply 450 V (approx. 70 V / cm) voltage to the platinum electrode connected to the buffer solution, and set the voltage at 0 V (Ground) to the ionic liquid containing the tip. An ionic liquid was injected into the tip by applying for 20 seconds and 5 seconds, respectively. In addition, the samples were sampled by applying voltage for 1 second and 0.1 second, respectively, to obtain two phase separated fragment samples of 300 pL and 30 pL. For multisampling, 0.1 μM riboflavin aqueous solution was injected into the tip by applying a voltage for 1 second each time.

7 to 9 show sampling schematics according to the present invention. 7 and 8 are photographs obtained by sampling samples having a volume of 300 pL and 30 pL, respectively, and FIG. 9 shows an image of a multi-sampled sample.

Since the sample injection method of the present invention uses a tapered capillary tip, a small amount of pL level can be injected, a sample of fL level can be sampled according to the degree of tapering, and two phase liquid substrates are used. No diffusion of the sample occurs after sampling.

Test Example

After completing the injection of 300 pL of 0.1 μM riboflavin aqueous solution sample using an electric field according to an embodiment of the present invention, the sample was placed in a laser guided fluorescence system within 30 seconds to measure a signal (FIG. 10 (a)). In addition, in order to compare the sensitivity of the signal of the sample of the present invention, the sample of 10 times (1 μM) dark concentration without using an ionic liquid under the same environmental conditions (the same voltage and time as the embodiment). Only 300 pL was injected into the tip for 1 second to measure the laser induced fluorescence signal (Fig. 10 (b)). As can be seen in Figure 10, when the ionic liquid was not used under the same conditions, despite the injection of a sample 10 times thicker concentration, the signal was hidden by the noise due to the diffusion could not observe the peak.

As such, when handling a very small sample of nL to pL or less, the signal is easily masked by noise due to the diffusion of the sample, it can be seen that this effect can be minimized by the analysis method of the present invention.

According to the present invention, the sample analysis method of injecting an ionic liquid by using an electric field before and after sample injection prevents the loss of the signal due to diffusion as much as possible, thereby providing excellent detection sensitivity and providing a high theoretical number of fractions. It is not only useful for detecting a variety of samples including micro-volume cells such as single-cell analysis in life science research, but also enables multi-segment sample injection with the same tip, which saves time and expense compared to conventional detection methods. It can be usefully used on lab-on-a-chip.

Claims (10)

A method of analyzing a sample by injecting a sample into a channel through a capillary tip into a microchip in which a channel is formed, and detecting the sample by fluorescence or electrochemical detection. A sample analysis method comprising sequentially injecting a mixed solution of a sample and a second ionic liquid not mixed with water using an electric field. The method of claim 1, The first and second ionic liquids are cationic ionic liquids or hexafluorophosphates, bis (trifluoromethylsulfonyl) imide, trifluoromethylsulfo selected from alkylimidazolium and N-alkylpyridinium. And an anionic liquid selected from nate and tetrafluoroborate. The method of claim 2, Sample method, characterized in that the alkyl group has 2 to 12 carbon atoms. The method of claim 1, Injecting the ionic liquid and the sample mixture solution by sequentially applying an electric field in the range of 1 to 600 V / cm for 0.1 seconds to 5 minutes to the mixed solution of the first and second ionic liquid or electrolyte aqueous solution and the sample , Sample analysis method. The method of claim 1, A method for analyzing a sample, characterized in that the inner wall of the capillary tip is coated with a cationic polymer. The method of claim 5, Wherein the cationic polymer is selected from polydimethyldiallyl ammonium chloride (PDDAC), polybrene (PB), polyethyleneimine (PEI), poly (methoxyethoxyethyl) ethylimine (PolyEG ₂ ) and polylysine (PL) A sample analysis method. The method of claim 1, The substrate of claim 1, wherein the substrate of the microchip is selected from rubber, silicone rubber, plastic, metal, glass and silica. The method of claim 1, And a capillary tip is of a material selected from rubber, silicone rubber, plastic, metal, glass and silica. The method of claim 1, A method for analyzing a sample, characterized in that the capillary tip is tapered. The method of claim 1, A sample analysis method, characterized in that the plurality of samples are injected in a segment in which the ionic liquid and the sample-containing mixed liquid are alternately repeated.

Images